The absorption of [14C]vitamin D3 and 25-[3H]hydroxyvitamin D3 into the mesenteric lymph and portal vein after simultaneous instillation in rat duodenum has been studied. The appearance of both isotopes in the mesenteric lymph and portal vein during the first 2 h was negligible. After 2-4 h the isotopes appeared both in the lymph and portal vein. The appearance of 25-[3H]hydroxyvitamin D3 in the mesenteric lymph was faster than that of [14C]vitamin D3. Total recovery in the lymph during 24 h for [14C]vitamin D3 and 25-[3H]hydroxyvitamin D3 was 15.2 and 14.3%, respectively. While 80% of vitamin D3 in mesenteric lymph was recovered in the chylomicrons (d less than 1.006 g/ml), 94% of 25-hydroxyvitamin D3 was recovered in the protein fraction with d greater than 1.21 g/ml. Seventy-one percent of 25-hydroxyvitamin D3 in the protein fraction was associated with the alpha-globulins. Both vitamin D3 and 25-hydroxyvitamin D3 that were recovered in the chylomicrons (d less than 1.006 g/ml) were transferred in vitro to the lymph fraction with d greater than 1.006 g/ml. Less than 12% of 25-hydroxyvitamin D3 was recovered in the liver after intravenous injection of mesenteric lymph labeled with 25-[3H]hydroxyvitamin D3. Previously, under similar conditions 40% of administered vitamin D3 was recovered in the liver.